HIF-1a/FOXO3a抑制DDR功能执行的分子机制及其在胃肠道间质瘤TKI继发耐药形成中的作用

Genomic instability, such as secondary mutations of c-kit/PDGFRA gene abnormal amplification, has been found to be the key driver for secondary resistance of tyrosine kinase inhibitor (TSR). Furthermore, more and more studies have confirmed that the inhibition of DNA damage response (DDR) is related to the development of genomic instability and indefinite cell proliferation. In our preliminary study, we found the increased expression of hypoxia-inducible factor-1a (HIF-1a) in TSR GIST samples, and HIF-1a could increase TSR clone and gene mutation frequency and decrease apoptosis. Importantly, HIF-1a and its downstream molecular FOXO3a could decrease the phosphorylation of several DDR related protein. Accordingly, we hypothesized a new mechanism for TSR: HIF-1a, through up-regulating the expression of FOXO3a, inhibits the function of activation and repair of DDR, thus resulting in genomic instability and TSR. To test this hypothesis, we will take GIST cell line as the carrier, and explore, step by step, the transcriptional regulation mechanism of HIF-1a on FOXO3a，and the protein and protein interaction between FOXO3a and DDR related proteins; additionally, by using in vitro and in vivo model, we will investigate the role of HIF-1a/FOXO3a/DDR pathway in the formation of TSR. Through this research, we will try to clarify the mechamisms of TSR and supply the experimental data for GIST therapy.

新研究表明c-kit/PDGFRA基因二次突变等基因组不稳定性是导致胃肠道间质瘤(GIST)酪氨酸激酶抑制剂继发耐药(TSR)的分子基础，并明确DNA损伤反应(DDR)是维护基因组稳定的基石。前期，我们在TSR临床标本中检测到缺氧诱导因子-1a(HIF-1a)高表达，并证实其能促进TSR克隆形成、减少细胞凋亡、增加基因突变频率；重要的是，HIF-1a及其下游分子FOXO3a抑制GIST细胞DDR功能执行蛋白磷酸化。据此我们提出TSR新机制：缺氧环境下，HIF-1a上调FOXO3a表达、抑制DDR损伤激活和修复功能执行，诱发GIST基因组不稳定，进而形成TSR。为验证该机制，本项目将逐步分析HIF-1a对FOXO3a、FOXO3a对DDR功能执行的作用机制；并通过TSR体内外模型，明确通路关键基因对TSR的调控作用。通过此研究，力图阐明TSR形成机制，为临床开展GIST新诊疗方案提供依据。

胃肠道间质瘤（Gastrointestinal stromal tumor, GIST）是胃肠道最常见的间叶源性肿瘤，其发病机制复杂。虽然当前研究表明其发病的关键分子机制为编码III型酪氨酸激酶受体的c-kit和血小板源性生长因子受体a基因突变，进而造成酪氨酸激酶持续激活，引发细胞无序增殖和凋亡抑制。但是该机制并不能完全解释GIST酪氨酸激酶抑制剂耐药和恶性生长。为此，在本研究中我们以HIF-1α为关键研究靶点，相继发现：(1)继发耐药标本中HIF-1α高表达，其能促进耐药克隆的形成。HIF-1α表达水平与肿瘤最大径、NIH危险度分级、WHO肿瘤分级、核分裂像计数、ki-67指数呈正相关，提示HIF-1α蛋白高表达与GIST的侵袭性生长相关。(2) GIST表达FOXO3a mRNA和蛋白，FOXO3a转录起始位点上游3000bp范围内存在4处缺氧反应元件（Hypoxia response element，HRE）结合位点。ChIP实验明确在缺氧环境下HIF-1α可结合于FOXO3a的启动子区。荧光素酶报告基因检测HIF-1α正向调控Foxo3a的promoter区。(3) HIF-1α、HIF-2α联合调控FOXM1的表达。生物信息学分析表明FOXM1启动子区存在可能存在5个HRE结合位点。ChIP实验明确在缺氧环境下HIF-1α可结合到定位于-25～-29的HRE1，；HIF-2α可以结合到FOXM1启动子区的HRE2（-301～-297）、HRE3（-303～-309）、HRE5（-2328～-2324）。FOXM1与GIST临床病理的相关性表明FOXM1蛋白表达水平与肿瘤最大径、ki-67指数、侵袭转移、NIH分级、WHO分级有显著的相关性。体外实验表明FOXM1促进GIST细胞生长、侵袭和转移。(4) 采用Oncomine分析，筛选出BMI1、STAT3、SHH、KLF4、SALL4、TERT在GIST中表达。免疫组织化学证实BMI1、TERT蛋白在GIST中阳性表达。BMI1与GIST临床病理的相关性表明BMI1核蛋白表达水平与与肿瘤体积、高有丝分裂计数、高Ki-67指数、高NIH分级和WHO分级相关。TERT则与GIST临床病理因素无明显的相关性。以上研究表明，缺氧-HIF-1α-FOXO3a/FOXM1信号通路在GIST继发耐药、恶性进展中的作用。